Tetralactam macrocycle based indicator displacement assay for colorimetric and fluorometric dual-mode detection of urinary uric acid.

An indicator displacement assay for colorimetric and fluorometric dual-mode detection of urinary uric acid (UA) was constructed using a water-soluble naphthalene-based tetralactam macrocycle and the phenoxazine dye, resorufin (RF). The visual detection of UA levels of volunteers was successfully realized using modified paper assays, which could be used for the home monitoring of urinary UA.

A highly sensitive sensor for colorimetric detection of palladium(II) in lysosomes and its applications.

Considering the scarcity of palladium ion probes with subcellular organelle targeting, especially probes with near-infrared (NIR) emission wavelength fluorophores, our group has been working to overcome this problem and looking forward to providing potential practical tools for exploring the toxicity of palladium ions at the subcellular level. In this paper, a novel colorimetric and NIR fluorescent probe, BHCy-Pd, for the specific detection of palladium ions (Pd2+) in lysosomes via an internal charge-transfer (ICT) mechanism was designed and synthesized. As expected, BHCy-Pd exhibited a rapid, selective, and sensitive response for palladium with an ultralow limit of detection at 5.9 nM, accompanied by a distinct color change from purple to blue. Furthermore, BHCy-Pd can be made into a simple test strip for rapid and easy detection of Pd2+ in practical applications. Importantly, BHCy-Pd is capable of specific distribution in lysosomes, and thus can detect Pd2+ in real-time, thereby providing a potential tool for studying the cytotoxicity of Pd2+ ions at the subcellular level.

Ratiometric fluorescence and colorimetric dual-mode sensing platform based on carbon dots for detecting copper(II) ions and D-penicillamine.

A sensing platform with both ratiometric fluorescence and colorimetric responses towards copper(II) ions (Cu2+) and D-penicillamine (D-pen) was constructed based on carbon dots (CDs). o-Phenylenediamine (OPD) was employed as a chromogenic development reagent for reaction with Cu2+ to generate the oxidation product 2,3-diaminophenazine (oxOPD), which not only emits green fluorescence at 555 nm, but also quenches the blue fluorescence of CDs at 443 nm via the inner filter effect (IFE) and Förster resonance energy transfer (FRET). Additionally, oxOPD exhibits obvious absorption at 420 nm. Since the intense chelation affinity of D-pen to Cu2+ greatly inhibits the oxidation of OPD, the intensity ratio of fluorescence at 443 nm to that at 555 nm (F443/F555) and the absorbance at 420 nm (A420) were conveniently employed as spectral response signals to represent the amount of D-pen introduced into the testing system. This dual-signal sensing platform exhibits excellent selectivity and sensitivity towards both Cu2+ and D-pen, with low detection limits of 0.019 µM and 0.092 µM, respectively. In addition, the low cytotoxicity of the testing reagents involved in the proposed sensing platform facilitates its application for live cell imaging.

Rapid fluorescent color analysis of copper ions on a smart phone via ratiometric fluorescence sensor.

A smartphone-assisted fluorescence color sensing system for rapid, convenient, and on-site detection of copper ions was developed. The ratiometric fluorescence sensor was fabricated by using silica-coated blue-light-emitting carbon dots and surface-grafted red-light-emitting cadmium-telluride quantum dots. After exposure to Cu2+ in 20 s, the red fluorescence was quenched obviously, while the blue fluorescence remained unchanged, and the sensor color changes continuously from red to blue under the ultraviolet lamp. The concentration (50-1200 nM) of copper ions could be measured by the fluorescence spectrum (excitation at 360 nm, dual-emission at 441 and 640 nm) with a detection limit of 7.7 nM. The fluorescence colors were converted to digital RGB values to calculate the concentration of copper ions by a smartphone with a detection limit of 9.6 nM. The method was applied to detecting copper ions spiked in real samples with recovery from 97.9 to 108.0% and RSD from 3.8 to 8.9%. Thus, this convenient and practical fluorescence color sensing system presents a new strategy for rapid, sensitive, and on-site determination of copper ions in environmental or biological samples.

Target-Responsive Smart Nanomaterials via a Au-S Binding Encapsulation Strategy for Electrochemical/Colorimetric Dual-Mode Paper-Based Analytical Devices.

Target-responsive nanomaterials attract growing interest in the application of drug delivery, bioimaging, and sensing due to the responsive releasing of guest molecules by the smart molecule gate. However, it remains a challenge to develop smart nanomaterials with simple assembly and low nonspecific leakage starting from encapsulation strategies, especially in the sensing field. Herein, Au nanoclusters (Au NCs) were first grown on porous carbon derived from ZIF-8 (PCZIF) to be employed as nanocarriers. By employing the Au NCs as linkers and aptamer (Apta) double-strand hybrids (target Apta and SH-complementary DNA) as capping units, we reported the novel target-responsive nanomaterials of Apta/Au NCs-PCZIF/hemin through Au-S binding encapsulation for sensing assays. The Au-S binding encapsulation strategy simplified the packaging procedure and reduced non-target responsive leakage. As a proof, ochratoxin A (OTA) as a model target participates in the double-strand hybrid competitive displacement reaction and triggered Apta conformation switches from a coil to a G-quadruplex structure accompanied by the dissociation of the gatekeeper. Simultaneously, the released hemin can initiate a self-assembly to form G-quadruplex/hemin DNAzyme. Interestingly, owing to DNAzyme providing electron transfer mediators and peroxidase-like activity, we proposed an electrochemical/colorimetric dual-mode paper-based analytical device (PAD) that provided self-verification to enhance reliability and accuracy, benefiting from independent signal conversion and transmission mechanism. As a consequence, the proposed dual-mode PAD could achieve sensitive electrochemical detection and visual prediction of OTA in the range of 1 pg/mL to 500 ng/mL and 50 pg/mL to 500 ng/mL, respectively. The electrochemical detection limit for OTA was as low as 0.347 pg/mL (S/N = 3). We believe that this work provides point-of-care testing (POCT) tools for a broad spectrum of applications.

Design of a High-Sensitivity Dimeric G-Quadruplex/Hemin DNAzyme Biosensor for Norovirus Detection.

G-quadruplexes can bind with hemin to form peroxidase-like DNAzymes that are widely used in the design of biosensors. However, the catalytic activity of G-quadruplex/hemin DNAzyme is relatively low compared with natural peroxidase, which hampers its sensitivity and, thus, its application in the detection of nucleic acids. In this study, we developed a high-sensitivity biosensor targeting norovirus nucleic acids through rationally introducing a dimeric G-quadruplex structure into the DNAzyme. In this strategy, two separate molecular beacons each having a G-quadruplex-forming sequence embedded in the stem structure are brought together through hybridization with a target DNA strand, and thus forms a three-way junction architecture and allows a dimeric G-quadruplex to form, which, upon binding with hemin, has a synergistic enhancement of catalytic activities. This provides a high-sensitivity colorimetric readout by the catalyzing H2O2-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline -6-sulfonic acid) diammonium salt (ABTS). Up to 10 nM of target DNA can be detected through colorimetric observation with the naked eye using our strategy. Hence, our approach provides a non-amplifying, non-labeling, simple-operating, cost-effective colorimetric biosensing method for target nucleic acids, such as norovirus-conserved sequence detection, and highlights the further implication of higher-order multimerized G-quadruplex structures in the design of high-sensitivity biosensors.

Portable on-chip colorimetric biosensing platform integrated with a smartphone for label/PCR-free detection of Cryptosporidium RNA.

Cryptosporidium, a protozoan pathogen, is a leading threat to public health and the economy. Herein, we report the development of a portable, colorimetric biosensing platform for the sensitive, selective and label/PCR-free detection of Cryptosporidium RNA using oligonucleotides modified gold nanoparticles (AuNPs). A pair of specific thiolated oligonucleotides, complementary to adjacent sequences on Cryptosporidium RNA, were attached to AuNPs. The need for expensive laboratory-based equipment was eliminated by performing the colorimetric assay on a micro-fabricated chip in a 3D-printed holder assembly. A smartphone camera was used to capture an image of the color change for quantitative analysis. The detection was based on the aggregation of the gold nanoparticles due to the hybridization between the complementary Cryptosporidium RNA and the oligonucleotides immobilized on the AuNPs surface. In the complementary RNA's presence, a distinctive color change of the AuNPs (from red to blue) was observed by the naked eye. However, in the presence of non-complementary RNA, no color change was observed. The sensing platform showed wide linear responses between 5 and 100 µM with a low detection limit of 5 µM of Cryptosporidium RNA. Additionally, the sensor developed here can provide information about different Cryptosporidium species present in water resources. This cost-effective, easy-to-use, portable and smartphone integrated on-chip colorimetric biosensor has great potential to be used for real-time and portable POC pathogen monitoring and molecular diagnostics.

Advanced graphene oxide-based paper sensor for colorimetric detection of miRNA.

MicroRNAs (miRNAs), found in blood and body fluids, have emerged as potential non-invasive biomarkers for disease and injury. miRNAs are quantitatively evaluated using typical RNA analysis methods such as the quantitative reverse transcription polymerase chain reaction, microarrays, and Northern blot, all of which require complex procedures and expensive reagents. To utilize miRNAs as practical biomarkers, it will be helpful to develop simple and user-friendly sensors. In this study, a paper-based miRNA sensor was developed by combining two methods: (1) target-recycled DNAzyme (Dz) amplification and (2) graphene oxide-assisted Dz blotting on paper. The Dz spots on paper caused a miRNA-dependent color change in presence of colorimetric reagents and facilitated the quantification of absolute amount of the target miRNA, irrespective of the volume, with high reproducibility. This approach is technologically straightforward and enables quantification of as low as 7.75 fmol miRNA using a portable smartphone.

Paper-based lateral flow assay using rhodamine B-loaded polymersomes for the colorimetric determination of synthetic cannabinoids in saliva.

Synthetic cannabinoids are one of the many substances of abuse widely spreading in modern society. Medical practitioners and law enforcement alike highly seek portable, efficient, and reliable tools for on-site detection and diagnostics. Here, we propose a colorimetric lateral flow assay (LFA) combined with dye-loaded polymersome to detect the synthetic cannabinoid JWH-073 efficiently. Rhodamine B-loaded polymersome was conjugated to antibodies and fully characterized. Two LFA were proposed (sandwich and competitive), showing a high level of sensitivity with a limit of detection (LOD) reaching 0.53 and 0.31 ng/mL, respectively. The competitive assay was further analyzed by fluorescence, where the LOD reached 0.16 ng/mL. The application of the LFA over spiked synthetic saliva or real human saliva demonstrated an overall response of 94% for the sandwich assay and 97% for the competitive LFA. The selectivity of the system was assessed in the presence of various interferents. The analytical performance of the LFA system showed a coefficient of variation below 6%. The current LFA system appears as a plausible system for non-invasive detection of substance abuse and shows promise for synthetic cannabinoid on-site sensing.

On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples.

This work modifies a loop-mediated isothermal amplification (LAMP) assay to detect the bovine respiratory disease (BRD) bacterial pathogens Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in a colorimetric format on a farm. BRD causes a significant health and economic burden worldwide that partially stems from the challenges involved in determining the pathogens causing the disease. Methods such as polymerase chain reaction (PCR) have the potential to identify the causative pathogens but require lab equipment and extensive sample processing making the process lengthy and expensive. To combat this limitation, LAMP allows accurate pathogen detection in unprocessed samples by the naked eye allowing for potentially faster and more precise diagnostics on the farm. The assay developed here offers 66.7-100% analytical sensitivity, and 100% analytical specificity (using contrived samples) while providing 60-100% concordance with PCR results when tested on five steers in a feedlot. The use of a consumer-grade water bath enabled on-farm execution by collecting a nasal swab from cattle and provided a colorimetric result within 60 min. Such an assay holds the potential to provide rapid pen-side diagnostics to cattle producers and veterinarians.

Colorimetric histology using plasmonically active microscope slides.

The human eye can distinguish as many as 10,000 different colours but is far less sensitive to variations in intensity1, meaning that colour is highly desirable when interpreting images. However, most biological samples are essentially transparent, and nearly invisible when viewed using a standard optical microscope2. It is therefore highly desirable to be able to produce coloured images without needing to add any stains or dyes, which can alter the sample properties. Here we demonstrate that colorimetric histology images can be generated using full-sized plasmonically active microscope slides. These slides translate subtle changes in the dielectric constant into striking colour contrast when samples are placed upon them. We demonstrate the biomedical potential of this technique, which we term histoplasmonics, by distinguishing neoplastic cells from normal breast epithelium during the earliest stages of tumorigenesis in the mouse MMTV-PyMT mammary tumour model. We then apply this method to human diagnostic tissue and validate its utility in distinguishing normal epithelium, usual ductal hyperplasia, and early-stage breast cancer (ductal carcinoma in situ). The colorimetric output of the image pixels is compared to conventional histopathology. The results we report here support the hypothesis that histoplasmonics can be used as a novel alternative or adjunct to general staining. The widespread availability of this technique and its incorporation into standard laboratory workflows may prove transformative for applications extending well beyond tissue diagnostics. This work also highlights opportunities for improvements to digital pathology that have yet to be explored.

A functional peptide-mediated colorimetric assay for mercury ion based on dual-modified gold nanoparticles.

In the work, a rapid and accurate biosensor for mercury ions (Hg2+) was constructed, with which aggregation of dual-modified (DGPFHR- and CALNN-) gold nanoparticles (D/C-AuNPs) could be triggered by the high specificity of peptides to Hg2+. The given peptide DGPFHR possesses great capability of capturing Hg2+, accompanied by the conformational folding. Under the circumstances, D/C-AuNPs were employed as the detection probes to accomplish the quantitative analysis of Hg2+. This is primarily because the specific Hg2+-induced folding of peptides reduces the electrostatic repulsion and steric hindrance, thus accelerating the AuNPs aggregation. The principle and application potential of this proposal was proved by evidence. And the results demonstrated that Hg2+ ions could be selectively detected as low as 28 nM with a linear range of 100-800 nM. In consideration of superior simplicity, selectivity, accuracy and stability, the protocol was advantageous over other projects in practical measurement of various water samples.

N, Cl-doped carbon dots for fluorescence and colorimetric dual-mode detection of water in tetrahydrofuran and development of a paper-based sensor.

N, Cl-doped carbon dots (N, Cl-CDs) were prepared by hydrothermal method from rhodamine B (RhB) and ethylenediamine (EDA). The resulting N, Cl-CDs exhibited fascinating solvent dependence and strict excitation independence. As the polarity of the solvent increased (from tetrahydrofuran (THF) to water), the emission spectrum of N, Cl-CDs was redshifted and the fluorescence efficiency decreased, which were attributed to hydrogen bond-induced aggregation. Taking advantage of these attributes, the N, Cl-CDs were used as suitable probes for fluorescence and colorimetric dual-mode detection of water in THF. The linear relationship was 0.5-100% water with the detection limit down to 0.093%. Moreover, the sensing platform was converted into a paper-based sensor for handy, real-time, and visible humidity sensing. N, Cl-CDs/PVA films were fabricated and realized continuously tunable solid-state fluorescence, further expanding their practical application.

Colorimetric cellulose-based test-strip for rapid detection of amyloid ß-42.

An innovative sensing assay is described for point-of-care (PoC) quantification of a biomarker of Alzheimer's disease, amyloid ß-42 (Aß-42). This device is based on a cellulose paper-dye test strip platform in which the corresponding detection layer is integrated by applying a molecularly imprinted polymer (MIP) to the cellulose paper surface. Briefly, the cellulose paper is chemically modified with a silane to subsequently apply the MIP detection layer. The imprinting process is confirmed by the parallel preparation of a control material, namely a non-imprinted polymer (NIP). The chemical changes of the surface were evaluated by Fourier transform infrared spectroscopy (FTIR), contact angle, and thermogravimetric analysis (TG). Proteins and peptides can be quantified by conventional staining methods. For this purpose, Coomassie blue (CB) was used as a staining dye for the detection and quantification of Aß-42. Quantitative determination is made possible by taking a photograph and applying an appropriate mathematical treatment to the color coordinates provided by the ImageJ program. The MIP shows a linear range between 1.0 ng/mL and 10 µg/mL and a detection limit of 0.71 ng/mL. Overall, this cellulose-based assay is suitable for the detection of peptides or proteins in a sample by visual comparison of color change. The test strip provides a simple, instrument-free, and cost-effective method with high chemical stability, capable of detecting very small amounts of peptides or proteins in a sample, and can be used for the detection of any (bio)molecule of interest.

Pandora box of BCA assay. Investigation of the accuracy and linearity of the microplate bicinchoninic protein assay: Analytical challenges and method modifications to minimize systematic errors.

Bicinchoninic colorimetric assay is very widely used for total protein quantitative analysis. We report that bicinchoninic (BCA) total protein assay linearity range and the assay sensitivity are counterbalancing factors. BCA assay true linear range may be considerably narrower than the 20-2000 µg/ml and therefore the choice of the assay calibration range should not solely be dictated by the general recommendations of the user guide, however by the test specific needs and subsequent assay quality control. Expanding the BCA assay range up to 2000 µg/ml comes together with unavoidable heavy negative biases at low protein concentrations. The negative bias at low protein concentration only exacerbates with longer incubation time and/or increased sample to working reagent ratio. To minimize the lack of accuracy at low protein concentration of a wide range BCA assay, we proposing an alternative approach: a two-step incubation and calibration. With a minimum of extra work, the two-step incubation/calibration approach is devoid of the standard BCA workflow disadvantages and biases.

TiO2 Nanotubes Alginate Hydrogel Scaffold for Rapid Sensing of Sweat Biomarkers: Lactate and Glucose.

Versatile sensing matrixes are essential for the development of enzyme-immobilized optical biosensors. A novel three-dimensional titanium dioxide nanotubes/alginate hydrogel scaffold is proposed for the detection of sweat biomarkers, lactate, and glucose in artificial sweat. Hydrothermally synthesized titanium dioxide nanotubes were introduced to the alginate polymeric matrix, followed by cross-linking nanocomposite with dicationic calcium ions to fabricate the scaffold platform. Rapid colorimetric detection (blue color optical signal) was carried out for both lactate and glucose biomarkers in artificial sweat at 4 and 6 min, respectively. The superhydrophilicity and the capillarity of the synthesized titanium dioxide nanotubes, when incorporated into the alginate matrix, facilitate the rapid transfer of the artificial sweat components throughout the sensor scaffold, decreasing the detection times. Moreover, the scaffold was integrated on a cellulose paper to demonstrate the adaptability of the material to other matrixes, obtaining fast and homogeneous colorimetric detection of lactate and glucose in the paper substrate when image analysis was performed. The properties of this new composite provide new avenues in the development of paper-based sensor devices. The biocompatibility, the efficient immobilization of biological enzymes/colorimetric assays, and the quick optical signal readout behavior of the titanium dioxide nanotubes/alginate hydrogel scaffolds provide a prospective opportunity for integration into wearable devices.

A fluorescent and colorimetric dual-channel sensor based on acid phosphatase-triggered blocking of internal filtration effect.

A colorimetric and fluorescent dual-channel detection method for acid phosphatase (ACP) activity has been constructed, based on the internal filtering effect between oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) and rhodamine B (RB). Au3+, which in situ form gold nanoparticles (AuNPs), can oxidize colorless 3,3',5,5'-tetramethylbenzidine (TMB) to oxTMB (blue color). The fluorescence of RB can be quenched by oxTMB due to the spectral overlap of emission of RB and absorption of oxTMB. By means of the above process, ACP can be determined because ACP promotes the hydrolysis of 2-phospho-L-ascorbic acid trisodium salt (AAP) to generate ascorbic acid (AA), which can inhibit the internal filtering effect between RB and oxTMB. No material preparation was needed for the determination of ACP. The colorimetric and fluorimetric methods can quantify ACP in the range 0.06-5.0 mU/mL and 0.03-5.0 mU/mL, respectively. Furthermore, a smartphone-assisted sensing platform has been constructed for on-site monitoring of ACP in the range 0.75-50 mU/mL, and the detection limit is 0.3 mU/mL. The methods developed can measure ACP in human serum successfully.

Development of a smartphone-based lateral-flow imaging system using machine-learning classifiers for detection of Salmonella spp.

Salmonella spp. are a foodborne pathogen frequently found in raw meat, egg products, and milk. Salmonella is responsible for numerous outbreaks, becoming a frequent major public-health concern. Many studies have recently reported handheld and rapid devices for microbial detection. This study explored a smartphone-based lateral-flow assay analyzer which employed machine-learning algorithms to detect various concentrations of Salmonella spp. from the test line images. When cell numbers are low, a faint test line is difficult to detect, leading to misleading results. Hence, this study focused on the development of a smartphone-based lateral-flow assay (SLFA) to distinguish ambiguous concentrations of test line with higher confidence. A smartphone cradle was designed with an angled slot to maximize the intensity, and the optimal direction of the optimal incident light was found. Furthermore, the combination of color spaces and the machine-learning algorithms were applied to the SLFA for classifications. It was found that the combination of L*a*b and RGB color space with SVM and KNN classifiers achieved the high accuracy (95.56%). A blind test was conducted to evaluate the performance of devices; the results by machine-learning techniques reported less error than visual inspection. The smartphone-based lateral-flow assay provided accurate interpretation with a detection limit of 5 × 104 CFU/mL commercially available lateral-flow assays.

Clinical Utility of Biosensing Platforms for Confirmation of SARS-CoV-2 Infection.

Despite collaborative efforts from all countries, coronavirus disease 2019 (COVID-19) pandemic has been continuing to spread globally, forcing the world into social distancing period, making a special challenge for public healthcare system. Before vaccine widely available, the best approach to manage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is to achieve highest diagnostic accuracy by improving biosensor efficacy. For SARS-CoV-2 diagnostics, intensive attempts have been made by many scientists to ameliorate the drawback of current biosensors of SARS-CoV-2 in clinical diagnosis to offer benefits related to platform proposal, systematic analytical methods, system combination, and miniaturization. This review assesses ongoing research efforts aimed at developing integrated diagnostic tools to detect RNA viruses and their biomarkers for clinical diagnostics of SARS-CoV-2 infection and further highlights promising technology for SARS-CoV-2 specific diagnosis. The comparisons of SARS-CoV-2 biomarkers as well as their applicable biosensors in the field of clinical diagnosis were summarized to give scientists an advantage to develop superior diagnostic platforms. Furthermore, this review describes the prospects for this rapidly growing field of diagnostic research, raising further interest in analytical technology and strategic plan for future pandemics.

Colorimetric ammonia (NH3) sensor based on an alginate-methylcellulose blend hydrogel and the potential opportunity for the development of a minced pork spoilage indicator.

Hydrogels based on alginate and methylcellulose were developed as a colorimetric indicator for monitoring minced pork spoilage. The hydrogel was fabricated by an external gelation method using Ca2+ as the crosslinking agent. The pH-sensitive dye bromothymol blue was incorporated into the hydrogel to act as an indicator. The hydrogel's swelling index increased with an increasing ratio of methylcellulose, suggesting that the water uptake capacity is tunable by the polymer composition. The hydrogel's compression strength is directly proportional to the alginate content. The hydrogel indicator demonstrated a color change from orange to yellow (day 6) upon detecting total volatile basic nitrogen (TVB-N) built up in the package during minced pork storage at 4 °C, and the results showed a positive correlation between the color change, TVB-N and pH change of minced pork. This result demonstrated the potential application of the hydrogel as a spoilage indicator in intelligent packaging.